This invention relates to a protein involved in the nuclear localization of proteins, and more specifically to a protein involved in maintaining circadian rhythms. The invention also relates to mutants of the protein, nucleic acid and amino acid sequences encoding the protein itself, as well as methods of using the protein including in drug assays.
Patterns of activity with periodicities of approximately 24 hours are termed circadian rhythms, and are governed by an internal clock that functions autonomously, but can be entrained by environmental cycles of light or temperature. These behaviors can be entrained to a xe2x80x9czeitgeiberxe2x80x9d (most commonly light), but are sustained under conditions of constant darkness and temperature, revealing activity of an endogenous biological clock. Circadian rhythms produced in constant darkness, for example, can also be reset by pulses of light. Such light pulses will shift the phase of the clock in different directions (advance or delay) and to varying degrees in a fashion that depends on the time of light exposure [Pittendrigh, in Handbook of Behavioral Neurobiology, 4, J. Aschoff, Ed., New York: Plenum, 1981, pp. 95-124].
Circadian rhythms appear to be a universal component of animal behavior [Pittendrigh, C. S., Proc. Natl. Acad. Sci USA, 58:1762-1767 (1967); Pittendrigh, C. S., Neurosciences, 437-458 (1974)]. Indeed, circadian physiological rhythms are not limited to the animal kingdom, and genetic screens have identified clock genes in Drosophila [Konopka and Benzer, Proc. Natl. Acad. Sci. USA, 68:2112-2116 (1971); and Sehgal et al., Science, 263:1603-1606 (1994)], Chlamydomonas [Bruce, V. G. Genetics, 70:537-548 (1972)], Neurospora [Feldman and Hoyle, Neurospora crassa Genetics, 75:605-613 (1973); Crosthwaite et al., Science, 276:763-769 (1997)], Cyanobacteria [Kondo et al., Science, 266:1233-1236 (1994)], Arabidopsis [Millar et al., Science, 267:1161-1163 (1995)], hamster [Ralph and Menaker, Science, 241:1225-1127(1988)], and mouse [Vitaterna et al., Science, 264:719-725 (1994)].
Fruit flies show circadian regulation of several behaviors [Pittendrigh in The Neurosciences Third Study Program, Chap. 38, F. O. Schmitt and F. G. Worden, Eds. (MIT Press, Cambridge Mass., 1974); Jackson, in Molecular Genetics of Biological Rhythms, pp. 91-121, M. W. Young, Ed. (Dekker, New York, 1993)]. When populations of Drosophila are entrained to 12 hours of light followed by 12 hours of darkness (LD 12:12), adults emerge from pupae (eclose) rhythmically, with peak eclosion recurring every morning. The eclosion rhythm persists when the entraining cues are removed and behavior is monitored in constant darkness, thus indicating the existence of an endogenous clock. Adult locomotor activity is also controlled by an endogenous clock and recurs rhythmically with a 24-hour period.
In the fruit fly Drosophila melanogaster, two genes are essential components of the circadian clock, period and timeless [Sehgal et al. Science 263:1603 (1994)]. Mutations in either of these genes can produce arrhythmicity or change the period of the rhythm by several hours [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971); Sehgal et al Science 263:1603 (1994)]. Molecular studies [Bargiello et al Proc. Natl. Acad. Sci. U.S.A. 81:2142 (1984); Reddy et al Cell 38:701 (1984); Myers Science 270:805 (1995); Hardin et al. Nature 343:536 (1990); Sehgal et al., Science, 270:808-810 (1995)] have shown that per and tim are transcribed with indistinguishable circadian rhythms that are influenced by an interaction of the TIM and PER proteins [Sehgal et al. Science 263:1603 (1994); Gekakiset et al Science, 270:811 (1995)]. A physical association of the two proteins appears to be required for accumulation and nuclear localization of PER [Sehgal et al Science 263:1603 (1994); Gekakiset et al. (1995); Price et al. EMBO J., 14:4044 (1995)]. It is likely that nuclear localization leads to suppression of per and tim transcription [Hardin et al. Nature, 343:536 (1990); Sehgal et al., Science, 270:808-810(1995)]. Cycles of gene expression are thought to be sustained by 5 hour differences in the phases of RNA and protein accumulation. The observed delays in PER accumulation may result, in part, from a requirement for TIM to stabilize PER [Sehgal et al Science 263:1603 (1994); Sehgal et al.Science, 270:808-810(1995); Price et al.EMBO J., 14:4044-4049 (1995)].
More specifically, mutations in the Drosophila period (per) gene, for example, disrupt circadian rhythms of pupal eclosion and adult locomotor behavior [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971)]. Although per has been cloned and sequenced and its pattern of expression has been analyzed [Baylies et al. in Molecular Genetics of Biological Rhythms, pp. 123-153, M. W. Young, Ed. (Dekker, New York, 1993); Rosbash and Hall Neuron 3:387 (1989)], the biochemical function of the PER protein is unknown. PER shares some homology with a family of transcription factors [Crews et al Cell 52:143 (1988); Nambu et al Cell 67:1157 (1991); Reisz-Porszasz et al Science 256:1193 (1992); Hoffman et al Cell 252:954 (1991); Burbach et al Proc. Natl. Acad. Sci. U.S.A. 89:8185 (1992)] that possess a common sequence motif called the PAS domain.
Immunocytochemical experiments demonstrated that PER is a nuclear protein in a variety of Drosophila tissues [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971); Baylies et al in Molecular Genetics of Biological Rhythms, pp. 123-153, M. W. Young, Ed. (Dekker, N.Y., 1993)]. In cells of the adult fly visual and nervous systems, the amount of PER protein fluctuates with a circadian rhythm [Edery et al Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994)], the protein is phosphorylated with a circadian rhythm [Edery et al., Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994)], and PER is observed in nuclei at night but not late in the day [Siwicki et al Neuron 1:141 (1988); Saez and Young Mol. Cell. Biol. 8:5378 (1988); Zerr et al J. Neurosci 10:2749 (1990)]. The expression of per RNA is also cyclic. However, peak mRNA amounts are present late in the day, and the smallest amounts are present late at night [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971)]. Three mutant allelesxe2x80x94perO, perS, and perL,xe2x80x94cause arrhythmic behavior or shorten or lengthen periods, respectively [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971)]. These mutations also produce corresponding changes in the rhythms of per RNA and protein amounts [Edery et al Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994); Hardin et al Nature 343:536 (1990); Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992); Sehgal et al Science 263:1603 (1994)] and PER immunoreactivity in nuclei [Sewicki et al Neuron 1:141 (1988); Saez and Young Mol. Cell. Biol. 8:5378 (1988); Zerr et al J. Neurosci. 10:2749 (1990)]. This suggests a possible role for molecular oscillations of per in the establishment of behavioral rhythms [Hardin et al, Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992)].
Several mutations that affect eclosion and locomotor activity have been isolated in behavioral screens [Jackson, in Molecular Genetics of Biological Rhythms, pp. 91-121, M. W. Young, Ed. (Dekker, N.Y., 1993); Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971); Rosbash and Hall Neuron 3:387 (1989); Baylies et al in Molecular Genetics of Biological Rhythms, pp. 123-153, M. W. Young, Ed. (Dekker, N.Y., 1993); Jackson, J. Neurogenet 1:3 (1983); Dushay et al J. Biol. Rhythms 4:1 (1989); Dushay et al Genetics 125:557 (1990); Konopka et al., Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1991)]. The best characterized, and those with the strongest phenotypes, are mutations at the X chromosome-linked period (per) locus [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971); Rosbash and Hall Neuron 3:387 (1989); Baylies et al in Molecular Genetics of Biological Rhythms, pp. 123-153, M. W. Young, Ed. (Dekker, N.Y., 1993); Jackson, J Neurogenet 1:3 (1983); Dushay et al J. Biol. Rhythms 4:1 (1989); Dushay et al Genetics 125:557 (1990)]. Missense mutations atper can lengthen or shorten the period of circadian rhythms, whereas null mutations abolish circadian rhythms altogether. The per gene is expressed in many cell types at various stages of development. In most cell types, the period protein (PER) is found in nuclei [James et al EMBO J. 5:2313 (1986); Liu et al Genes Dev. 2:228 (1988); Saez and Young Mol. Cell. Biol. 8, 5378 (1988); Liu et al J. Neurosci. 12:2735 (1992) Siwicki et al Neuron 1:141 (1988); Zerr et al J. Neurosci. 10:2749 (1990); Edery et al Proc. Natl. Acad. Sci. U.S.A. 91:2260 (1994)]. A domain within PER is also found in the Drosophila single-minded protein (SIM) and in subunits of the mammalian aryl hydrocarbon receptor [Crews et al Cell 52:143 (1988); Hoffman et al Science 252:954 (1991); Burbach et al Proc. Natl. Acad. Sci. U.S.A. 89:8185 (1992); Reyes et al Science 256:1193 (1992)], and this domain (PAS, for PER, ARNT, and SIM) mediates dimerization of PER [Huang et al Nature 364:259 (1993)]. The amounts of both PER protein and RNA oscillate with a circadian period, which is affected by the per mutations in the same manner as behavioral rhythms are affected [Siwicki et al Neuron 1:141 (1988); Zerr et al J. Neurosci 10:2749 (1990) Edery et al Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994); Hardin et al Nature 343:536 (1990); Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992)]. Given the homologies to sim and the aryl hydrocarbon receptor (which are thought to regulate transcription), the effects of per on behavioral rhythms have been postulated to depend on circadian regulation of gene expression, including that of per itself [Hardin et al, Nature 343:536 (1990); Hardin et al, Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992)].
Timeless is a second gene which has been associated with circadian rhythms in Drosophila [U.S. patent application Ser. No. 08/619,198 filed Mar. 21, 1996, hereby incorporated by reference in its entirety]. In the absence of the Timeless protein, TIM, gene products, such as the Period protein, PER, are not stable in the cytoplasm. Upon binding to the Timeless protein, proteins such as PER are stabilized and translocated into the nucleus. Once in the nucleus, the proteins act to inhibit the production of their own RNA. Both the tim and per genes are transcribed cyclically, and this transcription drives behavior. In particular, the gene products are present in the cytoplasm late in the day when a sleeping cycle is induced, while when the gene products are in the nucleus late at night, and a waking cycle follows.
The TIM protein not only acts as a nuclear translocation factor for the PER protein, but the PER protein also serves as a nuclear translocation factor for the TIM protein, thus indicating that PER and TIM act as mutual and reciprocal nuclear translocation factors. The nuclear translocation of the PER-TIM heterodimer is a crucial step in the regulation of both tim DNA and per DNA transcription.
The TIM protein also plays an important role in entraining the circadian rhythm of Drosophila, and by analogy other animals, to environmental cycles of light. This property of the TIM protein is due to its requirement for stabilizing the PER protein; its role in regulating per DNA transcription; and the TIM protein""s extreme sensitivity to light. Unlike the PER protein which requires the TIM protein for stability, the stability of the TIM protein is independent of the PER protein.
Our current understanding of the molecular regulation of circadian rhythmicity in Drosophila comes from integrating genetics and molecular biology. Null mutations in either of two genes, period (per) and timeless (tim), abolish behavioral rhythmicity, while alleles encoding proteins with missense mutations have been recovered at both loci and show either short- or long-period behavioral rhythms [Konopka and Benzer, Proc. Natl. Acad. Sci USA, 68:2112-2116 (1971); Sehgal et al., Science, 263:1603-1606 (1994); Rutila et al., Neuron, 17:921-929 (1996)]. The RNA and protein products of the genes oscillate with a circadian rhythm in wild-type flies. These molecular rhythms are abolished by null mutations of either gene, and the periods of all molecular rhythms are correspondingly altered in each long- and short-period mutant indicating a regulatory interaction between these genes (Hardin et al., Nature, 343:536-540 (1990); Edery et al., Proc. Natl. Acad. Sci USA, 91:2260-2264 (1994); Sehgal et al., Science, 263:1603-1606 (1994); Vosshall et al., Science, 263:1606-1609 (1994); Seghal et al., Science, 270:808-810 (1995); Price et al., EMBO J., 14:4044-4049 (1995); Hunter-Ensor et al., Cell, 84:677-685 (1996); Myers et al., Science, 271:1736-1740 (1996); Zeng et al., Nature, 380:129-135 (1996)].
Production of these molecular cycles appears to depend on the rhythmic formation and nuclear localization of a complex containing the PER and TIM proteins [Seghal et al., Science, 270:808-810 (1995); Gekakis et al., Science, 270:811-815 (1995); Lee et al., Science, 271:1740-1744 (1996); Saez and Young, Neuron, 17:911-920 (1996); Saez and Young, Neuron, 17:911-920 (1996)]. A physical interaction of PER and TIM is required for nuclear localization of either protein, and nuclear activity of these proteins coordinately regulates per and tim transcription through a negative feedback loop [Sehgal et al., Science, 263:1603-1606 (1994); Vosshall et al., Science, 263:1606-1609 (1994); Seghal et al., Science, 270:808-810 (1995); Gekakis et al., Science, 270:811-815 (1995); Hunter-Ensor et al., Cell, 84:677-685 (1996); Lee et al., Science, 271:1740-1744 (1996); Myers et al., Science, 271:1736-1740 (1996); Saez and Young, Neuron, 17:911-920 (1996); Zheng et al., Nature, 380:129-135 (1996)]. Studies of perL, a mutation that lengthens the period of behavioral rhythms [Konopka and Benzer, Proc. Natl. Acad. Sci. USA, 68:2112-2116 (1971)] and delays nuclear localization of PER protein [Curtin et al., Neuron, 14:365-372 (1995)], have shown that the PERL protein has reduced affinity for TIM [Gekakis et al., Science, 270:811-815 (1995). This suggests that rates of PER/TIM association influence the period of the molecular cycle in mutant and wild type flies.
Seghal et al., [Science, 270:808-810 (1995)] proposed a model for the Drosophila clock in which delayed formation of PER/TIM complexes ensures separate phases of per/tim transcription and nuclear function of the encoded proteins. Recent mathematical treatments of the Drosophila data are consistent with this model [Leloup and Goldbeter, J. Biol. Rhythms, 13:70-87 (1998)]. Entrainment of this oscillator is regulated through the TIM protein, which is rapidly eliminated from the nucleus and cytoplasm of pacemaker cells when Drosophila are exposed to daylight [Hunter-Ensor et al., Cell., 84:677-685 (1996); Lee et al., Science, 271:1740-1744 (1996); Myers et al., Science, 271:1736-1740 (1996); Zheng et al., Nature, 380:129-135 (1996)]. Studies of transgenic Drosophila have shown that adult behavioral rhythms can be linked to per and tim expression in a small group of central brain cells, the lateral neurons [Ewer et al., (1992); Frisch et al., Neuron, 12:555-570 (1994); Vosshall and Young, Neuron, 15:345-360 (1995)]. per and tim are also expressed in larval brain cells that are most likely the larval LNs [Kaneko et al., Neurosci., 17:6745-6760 (1997)], suggesting a basis for larval entrainment to light/dark cycles [Sehgal et al., Proc. Natl. acad. Sci. USA, 89:1423-1427 (1992)]. Oscillations of per and tim RNA, and PER and TIM proteins have been found outside of the head in a variety of tissues [Giebultowicz and Hege, Nature, 386:664 (1997); Emery et al., Proc. Natl. Acad. Sci. USA, 94:4092-4096 (1997); Plautz et al., Science, 278:1632-1635 (1997)]. Some of the latter oscillations were observed in vitro with isolated tissues, further indicating a cell autonomous mechanism [Giebultowicz and Hege, Nature, 386:664 (1997); Emery et al., Proc. Natl. Acad. Sci. USA, 94:4092-4096 (1997); Plautz et al., Science, 278:1632-1635 (1997)]. Mammalian homologues of per have recently been identified [Tei et al., Nature, 389 (1997); Sun et al., Cell, 90:1003-1011 (1997); Shigeyoshi et al., Cell, 91:1043-1053 (1997); Albrecht et al., Cell, 91:1055-1064 (1997); Shearman et al., Neuron, 19:1261-1269 (1997)], suggesting that the molecular basis of circadian rhythms may be conserved from flies to mammals. A related circadian oscillator has also been described at the molecular level in Neurospora through the detailed work of Dunlap and colleagues [reviewed by Dunlap et al., Annu. Rev. Genet., 30:579-601 (1996)].
Although key features of the Drosophila clock have been identified, the involvement of additional, essential factors is suspected from prior work. Since neither PER nor TIM has a recognizable DNA-binding motif, an unidentified transcription factor(s) should mediate repression in response to nuclear PER/TIM complexes [(reviewed by Rosbash et al., Harb. Symp. Quant. Biol., 76:265-278 (1996); Young et al., Harb. Symp. Quant. Biol., 61:279-284 (1996)]. PER fails to accumulate in the absence of TIM even in the presence of high per RNA levels [Vosshall et al., Neuron, 15:345-360 (1994); Price et al., EMBO J., 14:4044-4049 (1995)], indicating the existence of an activity that de-stabilizes cytoplasmic PER monomers. Both PER and TIM are phosphorylated with a circadian rhythm [Edery et al., Proc. Natl. Acad. Sci. USA, 94:4092-4096 (1994); Zeng et al., Nature, 380:129-135, 1996)] indicating unidentified kinases. PER, in particular, becomes progressively phosphorylated over many hours, and the timing of this is changed in period-altering mutants, leading to the suggestion that defined hyperphosphorylated form(s) of PER might signal PER degradation [Edery et al., Proc. Natl. Acad. Sci. USA, 94:4092-4096 (1994)].
In summary, circadian rhythms in Drosophila require periodic interaction of the PERIOD (PER) and TIMELESS (TIM) proteins. Physical associations of PER and TIM allow their nuclear translocation, and autoregulation of per and tim transcription through a negative feedback loop. Because PER/TIM heterodimers are only observed when high levels of per and tim RNA have accumulated, self-sustained oscillations are produced in the feedback loop [Gekakis et al., Science, 270:811-815 (1995); Hunter-Ensor et al., Cell, 84:677-685 (1996); Myers et al., Science, 271:1736-1740 (1996); Saez and Young, Neuron, 17:911-920 (1996); Sehgal et al., Science, 270:808-810 (1995) and Zeng et al, Nature, 380:129-135 (1996)]. Although molecular oscillations are maintained in constant darkness for per and tim RNA and for PER and TIM proteins, light can entrain the phases of these rhythms through rapid degradation of the light-sensitive TIM protein [Hunter-Ensor et al., Cell, 84:677-685 (1996); Myers et al., Science, 271:1736-1740 (1996) and Zeng et al., Nature, 380:129-135 (1996)]. Circadian oscillations of PER and TIM phosphorylation have also been described [Edery et al., PNAS, USA, 91:2260-2264 (1994) and Zeng et al., Nature, 380:129-135 (1996)]. However, prior studies have not demonstrated a function for these modifications. The recent identification of several PER homologues from mammals [Albrecht et al. Cell, 91:1055-1064 (1997); Shearman et al., Neuron, 19:1261-1269 (1997); Shigeyoshi et al., Cell, 19:1043-1053 (1997); Sun et al., Cell, 90:10031011 (1997) and Tei et al., Nature, 389:512-516 (1997)] suggests that, like many other biological processes, key molecules and mechanisms involved in circadian rhythms may be evolutionarily conserved between flies and mammals. A related mechanism has also been well defined in Neurospora [cf. Crosthwaite et al., Science, 276:763-769 (1997); Dunlap, Ann. Rev. of Gen, 30:579-601 (1996) and Garceau et al., Cell, 89:469-476 (1997)] and additional genes and proteins are known to play roles in the mouse [Antoch et al., (1997); King et al., Cell, 89:641-653 (1997) and Vitaterna et al., Science, 264:719-725 (1994)] the hamster [Ralph and Menaker, Science, 241:1225-1227 (1988)] and Arabidopsis [Millar et al., Science, 267:1161-1163 (1995)].
Therefore there is a need to identify other factors involved in circadium rythms. Furthermore, there is a need to use such factors to identify agents that can aid in the regulation of biological clocks, including as an aid in overcoming such maladies as jet lag.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The present invention discloses a protein DOUBLETIME (DBT), which is involved in circadium rhythms. More particularly, DBT, which is also known as casein kinase-1xcex5 (CK1xcex5) is shown herein to play a role in the regulation of the concentration of the important clock gene, Period (PER). The human CKIxcex5 has the nucleic acid sequence of SEQ ID NO:14, and the amino acid sequence of SEQ ID NO:9 [Fish, et al., J. Biol. Chem. 270:14875 (1995)]. Other CKIxcex5""s are exemplified in FIG. 18, below.
Therefore the present invention provides isolated nucleic acids and/or recombinant DNA molecules that encode DOUBLETIME (DBT) proteins, variants thereof, and the fragments thereof, including DBT (and DBT variants) chimeric peptides and proteins of the present invention. The present invention further provides the isolated and/or recombinant DBT proteins and fragments thereof including DBT (and DBT variants) chimeric peptides and proteins and DBT. In addition, the present invention provides antibodies to DBT and more specifically antibodies that are specific for the CK1xcex5 variant proteins. Methods of using these nucleic acids, proteins, and antibodies as reagents for drug screening and therapeutics are also provided.
One aspect of the present invention includes a nucleic acid that comprises a nucleotide sequence that encodes DBT or a DBT variant such as the tau CK1xcex5. In one such embodiment the nucleic acid comprises a nucleotide sequence that encodes a Drosophila DBT. In another embodiment, the nucleic acid comprises a nucleotide sequence that encodes a mammalian DBT. In a specific embodiment of this type the nucleic acid comprises a nucleotide sequence that encodes a tau hamster DBT having the amino acid sequence of SEQ ID NO:18. In a preferred embodiment the nucleic acid encoded a human variant of the DBT that corresponds/is the ortholog for the hamster tau CKIxcex5.
In a particular embodiment the nucleic acid comprises a nucleotide sequence that encodes a DBT having an amino acid sequence of SEQ ID NO:2. In a preferred embodiment of this type, the nucleic acid comprises the coding sequence of SEQ ID NO:1. In another embodiment the nucleic acid comprises a nucleotide sequence which encodes a DBT and hybridizes to the complementary strand of a nucleotide sequence that encodes a DBT having an amino acid sequence of SEQ ID NO:2. In a related embodiment the nucleic acid comprises a nucleotide sequence that encodes a DBT having an amino acid sequence of SEQ ID NO:2 comprising a conservative substitution. In another embodiment the nucleic acid comprises a nucleotide sequence which encodes a DBT and hybridizes to the complementary strand of a nucleotide sequence that encodes a DBT having an amino acid sequence of SEQ ID NO:2 comprising a conservative substitution.
In another embodiment the nucleic acid comprises a nucleotide sequence that encodes a DBT having an amino acid sequence of SEQ ID NO:16. In a preferred embodiment of this type, the nucleic acid comprises the coding sequence of SEQ ID NO:15. In another embodiment the nucleic acid comprises a nucleotide sequence which encodes a DBT and hybridizes to the complementary strand of a nucleotide sequence that encodes a DBT having an amino acid sequence of SEQ ID NO:16. In a related embodiment the nucleic acid comprises a nucleotide sequence that encodes a DBT having an amino acid sequence of SEQ ID NO:16 comprising a conservative substitution. In another embodiment the nucleic acid comprises a nucleotide sequence which encodes a DBT and hybridizes to the complementary strand of a nucleotide sequence that encodes a DBT having an amino acid sequence of SEQ ID NO:16 comprising a conservative substitution.
In an alternative embodiment the nucleic acid comprises a nucleotide sequence that encodes a DBT variant having an amino acid sequence of SEQ ID NO:3. In another such embodiment the nucleic acid comprises a nucleotide sequence that encodes a DBT variant having an amino acid sequence of SEQ ID NO:3 comprising a conservative substitution. In still another embodiment the nucleic acid comprises a nucleotide sequence that encodes a DBT variant having an amino acid sequence of SEQ ID NO:4. In yet another embodiment the nucleic acid comprises a nucleotide sequence that encodes a DBT variant having an amino acid sequence of SEQ ID NO:4 comprising a conservative substitution. In still another embodiment the nucleic acid comprises a nucleotide sequence that encodes a DBT variant having the amino acid sequence of SEQ ID NO:18. In yet another embodiment the nucleic acid comprises a nucleotide sequence that encodes a DBT variant having an amino acid sequence of SEQ ID NO:18 comprising a conservative substitution.
In addition the present invention provides nucleic acids encoding CK1xcex5 variants as defined below as dbtS# CK1xcex5, dbtL# CK1xcex5, and tau# CK1xcex5, and more specifically as dbtS CK1xcex5, a dbtL CK1xcex5, and a tau CK1xcex5. In addition the present invention provides nucleic acids that encode other variants of CK1xcex5, in particular, those that have one or more amino acid substitutions in the anion binding motif of the CK1xcex5, which in the hamster protein corresponds to Arg #178, Gly #215 and Lys #224 of SEQ ID NO:16 (see FIG. 18 below, for the analogous sites in other CK1xcex5""s).
In a related embodiment the present invention provides a nucleic acid that encodes an ATP-binding site of a DBT having an amino acid sequence of SEQ ID NO:6. In a preferred embodiment of this type, the nucleic acid comprises the coding sequence of SEQ ID NO:5. In another embodiment the nucleic acid encodes an ATP-binding site of a DBT having an amino acid sequence of SEQ ID NO:6 comprising a conservative substitution.
In another related embodiment the present invention provides a nucleic acid that comprises a nucleotide sequence that encodes a kinase catalytic domain of a DBT having an amino acid sequence of SEQ ID NO:8. In a preferred embodiment of this type, the nucleic acid comprises the coding sequence of SEQ ID NO:7. In another embodiment the nucleic acid comprises a nucleotide sequence that encodes a kinase catalytic domain of a DBT having an amino acid sequence of SEQ ID NO:8 comprising a conservative substitution.
The present invention further provides a nucleic acid consisting of at least 15, preferably at least 24, more preferably at least 36 consecutive nucleotides of a nucleotide sequence that encodes a DOUBLETIME protein having an amino acid sequence of SEQ ID NO:2. Another such embodiment is a nucleic acid consisting of at least 15, preferably at least 24, more preferably at least 36 consecutive nucleotides of a nucleotide sequence that encodes a DOUBLETIME protein having an amino acid sequence of SEQ ID NO:2 comprising a conservative substitution. In addition, the present invention provides nucleotide probes of at least 18 nucleotides for a nucleotide sequence that encodes a DOUBLETIME protein having an amino acid sequence of SEQ ID NO:2, and nucleotide probes of at least 12, preferably at least 18 and more preferably at least 27 nucleotides that are specific for a CK1xcex5, variant of the present invention.
All of the nucleic acids of the present invention can further comprise a heterologous nucleotide sequence. Furthermore, all of the nucleic acids of the present invention can be constructed into recombinant DNA molecules. Such recombinant DNA molecules can be operatively linked to an expression control sequence. The expression vectors containing the recombinant DNA molecules of the present invention are also provided by the present invention. In addition methods of expressing the recombinant DNA molecules for making the corresponding recombinant proteins and peptides are also provided. Thus, the recombinant proteins and peptides can be expressed in a cell (either a prokaryotic cell or a eukaryotic cell) containing an expression vector of the present invention by culturing the cell in an appropriate cell culture medium under conditions that provide for expression of the protein by the cell. In one such embodiment, a recombinant DOUBLETIME protein is expressed in a cell containing an expression vector of the present invention by culturing the cell in an appropriate cell culture medium under conditions that provide for expression of the DOUBLETIME protein by the cell. In a preferred embodiment of this type, the method further comprises the step of purifying the recombinant DOUBLETIME. The present invention also includes the recombinant DOUBLETIME proteins (including recombinant CK1xcex5 variants) and peptides made and/or purified by such methods.
Another aspect of the present invention is the protein encoded by a DOUBLETIME gene. In one embodiment the protein is a Drosophila DBT. In another embodiment, the protein is a mammalian DBT. In a preferred embodiment of this type the mammalian DBT is a human DBT. In a specific embodiment of this type the protein is a hamster enzyme. The present invention also provides the dbtS# CK1xcex5, dbtL# CK1xcex5, and tau# CK1xcex5 proteins and the unique fragments of these CK1xcex5 variant proteins. In a preferred embodiment of this type the protein is a human variant of the DBT that corresponds/is the ortholog for the hamster tau CKIxcex5.
In a particular embodiment the DBT has an amino acid sequence of SEQ ID NO:2. In another embodiment the DBT has an amino acid sequence of SEQ ID NO:2 comprising a conservative substitution. In another embodiment the DBT has an amino acid sequence of SEQ ID NO:16. In a related embodiment the DBT has an amino acid sequence of SEQ ID NO:16 comprising a conservative substitution. In a related embodiment the DBT is a DBT variant that has an amino acid sequence of SEQ ID NO:3. In another embodiment the DBT is a DBT variant that has an amino acid sequence of SEQ ID NO:3 comprising a conservative substitution. In still another embodiment the DBT variant has an amino acid sequence of SEQ ID NO:4. In yet another embodiment the DBT variant has an amino acid sequence of SEQ ID NO:4 comprising a conservative substitution. In another embodiment the DBT has an amino acid sequence of SEQ ID NO:18. In a related embodiment the DBT has an amino acid sequence of SEQ ID NO:18 comprising a conservative substitution.
In a related embodiment the present invention provides a protein or peptide comprising an ATP-binding site of a DBT that has an amino acid sequence of SEQ ID NO:6. In another embodiment the protein or peptide comprises an ATP-binding site of a DBT having an amino acid sequence of SEQ ID NO:6 comprising a conservative substitution. In another related embodiment the present invention provides a protein or peptide comprising a kinase catalytic domain of a DBT having an amino acid sequence of SEQ ID NO:8. In another embodiment the protein or peptide comprises a kinase catalytic domain of a DBT that has an amino acid sequence of SEQ ID NO:8 comprising a conservative substitution.
The present invention further provides fragments of the proteins and peptides of the present invention, including proteolytic fragments. In one embodiment the proteolytic fragment comprises at least six amino acids. In a preferred embodiment the proteolytic fragment comprises at least nine amino acids. In a more preferred embodiment the proteolytic fragment comprises at least fifteen amino acids. In one particular embodiment the fragment binds to the period (PER) protein. In another particular embodiment the fragment has protein kinase activity.
The present invention further provides fusion proteins comprising the proteins, peptides and fragments thereof of the present invention. Thus all of the DBTs (and variant DBTs) and fragments thereof of the present invention can be modified, placed in a fusion of chimeric peptide or protein, or labeled e.g., to have an N-terminal FLAG-tag, or a S.Tag. In a particular embodiment a DBT can be modified to contain a marker protein such as green fluorescent protein as described in U.S. Pat. No. 5,625,048 filed Apr. 29, 1997 and WO 97/26333, published Jul. 24, 1997 the disclosures of which each are hereby incorporated by reference herein in their entireties.
Still another aspect of the present invention provides an antibody to a DBT (or variant DBT) or fragment thereof, of the present invention. In a particular embodiment the antibody is specific for the CK1xcex5 variants of the present invention. In one embodiment the antibody of is a polyclonal antibody. In another embodiment the antibody is a monoclonal antibody. In a particular embodiment of this type the monoclonal antibody is a chimeric antibody. The present invention further provides immortal cell lines that produce the monoclonal antibodies of the present invention.
The present invention further provides CK1xcex5 (or CK1xcex5 variant) transgenic, knockin, and knockout animals. These animals can be used as animal models in drug screening assays for drugs that can treat sleep disorders or jet lag. Indeed, all of the CK1xcex5 variants disclosed herein can be used in the transgenic or knockin animals. In a preferred embodiment the transgenic or knockin animal is a mouse. In a particular embodiment the mouse is a tau# CK1xcex5 knockin mouse. In a preferred embodiment of this type the mouse is a tau CK1xcex5 knockin mouse. In another embodiment the mouse is a dbtS# CK1xcex5 knockin mouse. In a preferred embodiment of this type the mouse is a dbtS CK1xcex5 knockin mouse. In still another embodiment the mouse is a dbtL# CK1xcex5 knockin mouse. In a preferred embodiment of this type the mouse is a dbtL CK1xcex5 knockin mouse. The phenotypes of these mice preferably correspond to that found for the prior Drosophila and hamster variants.
The cells from the knockin and transgenic animals of the present invention and cells that are constructed to contain a variant CK1xcex5 are also part of the present invention. These cells can also be used in the drug assays described below.
Another aspect of the present invention provides methods for detecting the presence or activity of the DBTs of the present invention. One such embodiment comprises contacting a biological sample from an organism in which the presence or activity of the DBT is suspected with a binding partner for the DBT under conditions that allow binding of the DBT to the binding partner to occur, and then detecting whether the binding has occurred between the DBT and the binding partner in the sample. Detection of such binding indicates that the DBT is present in the sample.
A related aspect of the present invention includes methods for detecting a casein kinase1xcex5 (CK1xcex5) variant in a mammal. In a particular embodiment of this type the mammal has a sleep or related disorder. Preferably the mammal is a human. One method comprises obtaining a biological sample from the mammal and assaying the biological sample for the presence of a CK1xcex5variant. In another embodiment the biological sample is assayed for the presence of a nucleic acid encoding the CK1 variant. Preferably the CK1xcex5 variant is a CK1xcex5 polymorphism. In one particular embodiment the CK1xcex5 variant is a dbtS# CK1xcex5. In a preferred embodiment of this type the CK1xcex5 variant is a dbtS CK1xcex5. In another particular embodiment the CK1xcex5 variant is a dbtL# CK1xcex5. In a preferred embodiment of this type the CK1xcex5 variant is a dbtL CK1xcex5. In yet another particular embodiment the CK1xcex5 variant is a tau# CK1xcex5. In a preferred embodiment of this type the CK1xcex5 variant is a tau CK1xcex5.
In a particular embodiment the biological sample is assayed by being contacted with a probe that specifically binds to the nucleic acid that encodes the CK1xcex5 variant under conditions that allow the binding of the probe to the nucleic acid. The assaying can either be performed in vitro, after making a cell extract or in situ using FISH for example. In a particular embodiment of this type the probe is a nucleotide probe, and preferably the assaying is performed by hybridizing a labeled probe to the nucleic acid encoding the CK1xcex5 variant that is present in the biological sample. In another embodiment the probe is a primer, and the assaying includes performing PCR on the nucleic acid encoding the CK1xcex5 variant that is present in the biological sample. Alternatively, a probe can specifically bind to the CK1xcex5 variant present in the biological sample. In one such embodiment the probe is an antibody. In this case the antibody can either be detected directly or indirectly. In any case, the binding of the probe to the CK1xcex5 variant or the nucleic acid that encodes it, facilitates the detection of the CK1xcex5 variant in the biological sample. Alternatively, a CK1xcex5 variant can be detected through determining its enzymatic activity.
Once a CK1xcex5 variant is detected the nucleotide sequence of the nucleic acid encoding the CK1xcex5 variant can be determined. The isolated nucleic acid is also part of the present invention. In a particular embodiment the method can further comprise constructing a recombinant DNA that encodes the coding region of the nucleic acid. In addition, the recombinant DNA can be placed into an expression vector and expressed to make the corresponding recombinant protein. The isolated recombinant protein is also part of the present invention. The recombinant protein can also be assayed for its kinase and/or binding activity towards one or more variants of PER.
Still another aspect of the present invention includes methods of identifying an agent that modulates the interaction between a PER protein and a DBT protein. One such method comprises contacting the PER protein with the DBT protein in the presence of the agent under conditions in which the DBT protein binds the PER protein in the absence of the agent. The binding of the PER protein and the DBT protein is then measured. An agent that modulates the interaction between the PER protein and the DBT protein is identified when the binding of the PER protein with the DBT protein is different in the presence of the agent than in its absence. An agent that modulates the PER protein-DBT protein interaction by enhancing the binding is identified as an agonist. On the other hand, an agent that modulates the PER protein-DBT protein interaction by diminishing the binding is identified as an antagonist. In a preferred embodiment the DBT protein is human casein kinase Ixcex5.
In a particular embodiment, the step of determining the binding of the PER protein and the DBT protein includes determining the dissociation constant between the PER protein and the DBT protein. In another embodiment, an active fragment of the PER protein is used in place of the PER protein. In this case the active fragment of the PER protein minimally retains its DBT protein binding capacity. In a related embodiment, an active fragment of the DBT protein is used in place of the DBT protein. In this case the active fragment of the DBT protein minimally retains its PER protein binding capacity. In still another embodiment, an active fragment of the PER protein is used in place of the PER protein and an active fragment of the DBT protein is used in place of the DBT protein. Again, the active fragment of the PER protein minimally retains its DBT protein binding capacity, whereas the active fragment of the DBT protein minimally retains its PER protein binding capacity.
A related aspect of the present invention includes a method of identifying an agent that modulates the phosphorylation of a PER protein by a DBT. One such embodiment comprises contacting the PER protein with the DBT protein in the presence of the agent under conditions in which the DBT protein phosphorylates the PER protein in the absence of the agent. The phosphorylation of the PER protein is then measured (either directly or indirectly). An agent that modulates the phosphorylation of the the PER protein by the DBT protein is identified when the phosphorylation of the PER protein is different in the presence of the agent than in its absence. An agent that modulates the DBT-dependent phosphorylation of the PER protein by enhancing the phosphorylation is identified as an agonist. On the other hand, an agent that modulates the DBT-dependent phosphorylation of the PER protein by diminishing the phosphorylation is identified as an antagonist. In a preferred embodiment the DBT protein is human casein kinase Ixcex5.
In a particular embodiment, an active fragment of the PER protein is used in place of the PER protein. In this case the active fragment of the PER protein minimally retains its phosphorylation site, but preferably also retains its DBT protein binding capacity. In a related embodiment, an active fragment of the DBT protein is used in place of the DBT protein. In this case the active fragment of the DBT protein minimally retains its kinase activity but preferably also retains its PER protein binding capacity. In still another embodiment, an active fragment of the PER protein is used in place of the PER protein and an active fragment of the DBT protein is used in place of the DBT protein. Again, the active fragment of the PER protein minimally retains its phosphorylation site, but preferably also retains its DBT protein binding capacity, whereas the active fragment of the DBT protein minimally minimally retains its kinase activity but preferably also retains its PER protein binding capacity.
Such assays can be performed in vitro or in situ (for example in cells as in the cis-trans assays as described below). In one such in situ assay, the proteins or active fragments thereof are contacted in the cell by expressing the corresponding nucleic acids that encode the CK1xcex5 and PER in the cell. In addition, any of the CK1xcex5 variants of the present invention can be used in place of the wild type CK1xcex5 in the assays. Additional assays are also exemplified in Example 3, below.
The present invention also provides methods of using the transgenic and knockin animals of the present invention in drug assays and screens. More specifically, nucleic acids that express the various CK1xcex5 variants can be used to construct knockin mice which express the corresponding CK1xcex5 variant. In a particular embodiment the knockin mouse encodes a dbtS# CK1xcex5. In another embodiment the knockin mouse encodes a dbtL# CK1xcex5. In yet another embodiment the knockin mouse encodes a tau# CK1xcex5. The knockin mice can then be used in in vivo drug assays. Similarly cells obtained from the knockin mice and cells constructed to comprise a CK1xcex5 variant can also be employed in such assays.
In a particular embodiment an agent is administered to a CK1xcex5 variant knockin mouse having a specific phenotype due to the expression of the CK1xcex5 variant. The effect of the agent on the phenotype is then determined. An agent that modifies the specific phenotype is then selected. Agents can thus be identified which either extend or contract the animal""s period of rhythm. In one embodiment the knockin mouse has a shorter period of rhythm, e.g., a dbtS CK1xcex5, or a tau CK1xcex5, whereas in another embodiment the knockin mouse has a longer period of rhythm, e.g., the dbtL CK1xcex5.
Alternatively, the agents can be administered to the knockin or transgenic animal but the assay can be performed in situ or in vitro. Since CK1xcex5 is known to be a kinase, its relative activity is readily measured (see Examples below). Furthermore, since PER is a disclosed substrate for CK1xcex5, binding assays (including affinity kinase assays, see Example 3) with PER can also be performed. In addition, when the drug assay is performed in vivo, assays that measure locomotor activity (as described in Example 3) can be employed.
Yet another aspect of the present invention comprises methods of identifying the nucleotide and amino acid sequences of homologues to the Drosophila doubletime gene. Once the coding region of the nucleotide sequence is identified, the corresponding amino acid sequence can be readily determined using the genetic code, preferably with the aid of a computer. Preferably the full-length nucleotide sequence of the coding region of a homologue to the Drosophila doubletime gene is identified. Recombinant DNA molecules and the recombinant DBT proteins obtained by these methods are also part of the present invention.
One method of identifying a nucleotide sequence of the coding region of a homologue to the Drosophila doubletime gene comprises comparing SEQ ID NO:2 with the amino acid sequences encoded by nucleic acids that are obtained from a library of nucleic acids containing partial nucleotide sequences of the coding regions from non-Drosophila genes. Preferably this determination is aided by computer analysis. A nucleic acid containing a partial nucleotide sequence of a coding region from a non-Drosophila gene that is highly homologous to SEQ ID NO:2 can then be selected. Methods of ascertaining which nucleic acid and amino acid sequences are highly homologous are described below.
The full-length sequence of the coding region of the non-Drosophila gene is preferably determined. The sequence is identified as being that of the homologue to the Drosophila doubletime gene when it is highly homologous to SEQ ID NO:2 as discussed below. In a preferred embodiment this method further comprises determining whether the nucleotide sequence that contains a coding region for an amino acid sequence that is highly homologous to SEQ ID NO:2 is also expressed in the corresponding suprachiasmatic nucleus (SCN), i.e., if the putative homologue is a mouse homologue, the SCN of a mouse is tested [Sun et al., Cell 90:1003-1011 (1997)]. When the nucleotide sequence is expressed in the SCN, it is identified as the nucleotide sequence of the coding region of the homologue to the Drosophila doubletime gene. One means of determining whether the nucleotide sequence is expressed in the SCN is through the use of a labeled nucleotide probe for the nucleotide sequence that contains the coding region for an amino acid sequence that is highly homologous to SEQ ID NO:2. The labeled nucleotide probe can then be hybridized with a sample containing nucleic acids that are expressed in the SCN under stringent conditions. If hybridization is detected, the sequence is identified as being that of the homologue to the Drosophila doubletime gene. Similarly, a PCR primer can be used to aid in the confirmation of the identification of a nucleotide sequence of the coding region of the homologue to the Drosophila doubletime gene
In a particular embodiment of the method, determining the full-length sequence of the coding region is performed by sequencing the insert of a plasmid which contains a nucleic acid encoding an amino acid sequence that is highly homologous with SEQ ID NO:2. In this case, the insert comprises the nucleic acid. In another embodiment, the full-length sequence is determined by PCR.
A related embodiment includes a method of identifying the full-length nucleotide sequence of the coding region of a homologue to the Drosophila doubletime gene that comprises determining the percent homology of SEQ ID NO:2 to amino acid sequences encoded by nucleotide sequences from a library of nucleotide sequences for non-Drosophila genes and then selecting a nucleotide sequence that contains a coding region for an amino acid sequence that is highly homologous to SEQ ID NO:2. The full-length nucleotide sequence of the coding region for the amino acid sequence is determined and the full-length nucleotide sequence of the coding region of the homologue to the Drosophila doubletime gene is identified. This method can also comprise determining whether the nucleotide sequence is expressed in the suprachiasmatic nucleus (SCN). When the nucleotide sequence is expressed in the SCN, it is identified as the nucleotide sequence of the coding region of the homologue to the Drosophila doubletime gene.
In another embodiment, the method can further comprise constructing a recombinant DNA that contains the coding region. In one such embodiment a recombinant DBT protein is made by expressing the recombinant DNA. In a preferred embodiment of this type an activity of the recombinant Drosophila DBT is assayed. In one such embodiment, the activity assayed is the ability of the recombinant protein to bind to PER. In another such embodiment the activity assayed is the ability of the recombinant protein to act as a protein kinase. In yet another such embodiment the activity assayed is the ability of the recombinant protein to phosphorylate PER. The sequence is identified as being that of the homologue to the Drosophila doubletime gene when the recombinant protein has the activity of the Drosophila DBT.
In addition the present invention provides test kits for demonstrating the presence or absence of a DBT or a DBT variant in a cellular sample. One such kit comprises a predetermined amount of a binding partner of the protein or the nucleic acid that encodes the DBT or DBT variant. Preferably the binding partner is detectably labeled. A preferred kit further comprises a predetermined amount of the DBT and/or DBT variant to be used as a standard. A kit of the present invention can also provide a protocol for using the kit.
The present invention also provides methods of preventing and/or treating disorders of a circadian rhythm which include depression, narcolepsy and jet lag. Such methods rely on temporary antagonisms to transiently inhibit the natural clock, and then supplying agonists to subsequently reset it e.g., for the treatment of jet lag. One such embodiment comprises administering to an animal a therapeutically effective amount of a DBT. Another such embodiment comprises administering to an animal a therapeutically effective amount of an agent capable of promoting the production and/or activity of a DBT. Yet another such embodiment comprises a mixtures of such agents. Still another embodiment comprises administering to an animal a therapeutically effective amount of an agent capable of inhibiting the activity of the DBT.
Accordingly, it is a principal object of the present invention to provide DBTs in purified form that exhibit activities associated with circadium rhythms.
It is a further object of the present invention to provide antibodies to the DBTs, and methods for their preparation, including by recombinant means.
It is a still further object of the present invention to provide nucleic acids encoding DBTs.
It is a further object of the present invention to provide a method for detecting the presence of the DBT in mammals.
It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, that are potentially effective in either mimicking the activity or combating the adverse effects of the DBTs in mammals.
It is a still further object of the present invention to provide a method for the treatment of mammals to control depression, jet lag and/or narcolepsy.
It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon DBT or upon agents or drugs that control the production, or that mimic or antagonize the activities of the DBT.
It is a still further object of the present invention to provide a method of obtaining a human homologue to the Drosophila DBT.
It is a still further object of the present invention to provide Drosophila expressing mutant (variant) forms of DBT.
It is a still further object of the present invention to provide transgenic or knockin animals that express mutant (variant) forms of DBT.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.